Báo cáo sinh học: "Protein kinase CK2a is overexpressed in colorectal cancer and modulates cell proliferation and invasion via regulating EMT-related genes" docx

Zou et al Journal of Translational Medicine 2011, 9:97 http://www.translational-medicine.com/content/9/1/97 RESEARCH Open Access Protein kinase CK2a is overexpressed in colorectal cancer and modulates cell proliferation and invasion via regulating EMT-related genes Jinjin Zou1, Hesan Luo1, Qin Zeng1, Zhongyi Dong1, Dehua Wu1* and Li Liu2* Abstract Background: Protein kinase CK2 is a highly conserved, ubiquitous protein serine/threonine kinase that phosphorylates many substrates and has a global role in numerous biological and pathological processes Overexpression of the protein kinase CK2a subunit (CK2a) has been associated with the malignant transformation of several tissues, with not nearly as much focus on the role of CK2a in colorectal cancer (CRC) The aims of this study are to investigate the function and regulatory mechanism of CK2a in CRC development Methods: Expression levels of CK2a were analyzed in 144 patients (104 with CRC and 40 with colorectal adenoma) by immunohistochemistry Proliferation, senescence, motility and invasion assays as well as immunofluorescence staining and western blots were performed to assess the effect of CK2a in CRC Results: The immunohistochemical expression of nuclear CK2a was stronger in tumor tissues than in adenomas and normal colorectal tissues Suppression of CK2a by small-interfering RNA or the CK2a activity inhibitor emodin inhibited proliferation of CRC cells, caused G0/G1 phase arrest, induced cell senescence, elevated the expression of p53/p21 and decreased the expression of C-myc We also found that knockdown of CK2a suppressed cell motility and invasion Significantly, CK2a inhibition resulted in b-catenin transactivation, decreased the expression levels of vimentin and the transcription factors snail1 and smad2/3, and increased the expression of E-cadherin, suggesting that CK2a regulates the epithelial-mesenchymal transition (EMT) process in cancer cells Conclusions: Our results indicate that CK2a plays an essential role in the development of CRC, and inhibition of CK2a may serve as a promising therapeutic strategy for human CRC Introduction Colorectal cancer (CRC) is the second-most common cause of cancer death in the West [1] and its incidence in China has increased rapidly during the past few decades [2] Colorectal cancers can be divided into tumors exhibiting chromosomal instability and tumors exhibiting microsatellite instability [3,4] In the last few years, molecular biology advances have led to a growing knowledge of the mechanisms underlying CRC development, including the mutational activation of oncogenes * Correspondence: wudehua.gd@gmail.com; liliu.gd@gmail.com Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China Hepatology Unit and Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China Full list of author information is available at the end of the article and alteration of several tumor suppressor genes, such as adenomatous polyposis coli (APC), deleted in colorectal cancer (DCC) and p53 [5-8] However, molecular markers that indicate the occurrence and development of CRC are still needed Protein kinase CK2 (formerly casein kinase II) has traditionally been classified as a messenger-independent protein serine/threonine kinase that is typically found in tetrameric complexes consisting of two catalytic (a and/ or a’) subunits and two regulatory b subunits [9] To date, more than 300 CK2 substrates have been identified; one third of these are implicated in gene expression and protein synthesis as translational elements [10] CK2aknockout mice are not viable because of defects in heart and neural tube development [11] The disruption of CK2a expression in Saccharomyces cerevisiae and knockout of CK2b in mice are lethal events, indicating the © 2011 Zou et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Zou et al Journal of Translational Medicine 2011, 9:97 http://www.translational-medicine.com/content/9/1/97 importance of CK2 in the maintenance of cell viability during the normal cell life and embryogenesis [12,13] CK2a also participates in the regulation of various cell cycle stages, presumably through phosphorylation of the proteins associated with cell cycle progression [14] Furthermore, CK2 involvement has been found in chromatin remodeling as well as protein transcription, translation, and degradation [15-17] Recent studies suggest that CK2 creates an environment that is favorable for the development of the tumor phenotype [18] In the present study, we assessed CK2a expression in colorectal cancer, adenoma, and normal colorectal epithelium and found CK2a involvement in CRC tumorigenesis Moreover, the role of CK2a in cell proliferation, senescence, motility and invasion was examined in CRC cell lines that were subjected to CK2a knockdown or to the CK2a activity inhibitor emodin Further analysis was conducted to elucidate the mechanisms of CK2a involvement in the occurrence and development of CRC Page of 11 Table Clinicopathological characteristics of the 104 patients and expression of CK2a in CRC N (%) Gender Male 56 (53.8) Female 48 (46.2) ≥55 54 (51.9) 0.05) Because T describes how far the main (primary) tumor has grown into the wall of the intestine and whether it has grown into nearby areas, we speculated that CK2a may participate in CRC cell invasion Figure Immunohistochemical detection of CK2a expression in colorectal cancers, adenomas and adjacent normal colorectal mucosa Staining was (A) negative in normal colorectal epithelium cells, (B, C) weak to moderate in the nuclei of colorectal adenoma cells, (D, E, F) and strong in the nuclei of colorectal cancer cells (E is a close-up of the inset in D [framed in red]) Original magnification: × 200 (D), × 400 (A, B, C, E, F) Zou et al Journal of Translational Medicine 2011, 9:97 http://www.translational-medicine.com/content/9/1/97 Page of 11 Figure CK2a protein expression in CRC tissues and cell lines (A) Western blot analysis of CK2a expression in eight pairs of CRC tissues and adjacent, normal colorectal mucosa tissues N: normal colorectal mucosa tissue; T: tumor tissue (B) Quantitative analysis of CK2a protein expression in eight pairs of CRC tissues and adjacent normal colorectal mucosa tissues Columns, mean CK2a protein level after normalizing the data to GAPDH expression; bars, SD *P < 0.01 (C) Western blot was used to detect CK2a expression in five CRC cell lines GAPDH expression was used as a loading control CK2a regulates growth, proliferation and senescence of CRC cell lines Because the process of tumorigenesis is closely correlated with eternal proliferation of tumor cells, we determined whether CK2a expression plays a role in human CRC cell growth and proliferation using siRNA to knock down CK2a expression or emodin to inhibit CK2a activity (Figure 3A) The MTT assay showed that knockdown of CK2a significantly decreased CRC cell proliferation compared to the control (nonspecific siRNA) (F = 32.854, P < 0.01 for LoVo cells; F = 32.655, P < 0.01 for SW480 cells), and treatment with emodin markedly reduced proliferation (F = 33.290, P < 0.01 for LoVo cells; F = 57.052, P < 0.01 for SW480 cells; Figure 3B) Furthermore, in the colony formation assay, inhibition of CK2a expression dramatically decreased the number of CRC colonies (t = 20.252, P < 0.01 for LoVo cells; t = 12.034, P < 0.01 for SW480 cells; Figure 3C) and promoted CRC cell senescence (t = 43.052, P < 0.01; Figure 3D) Taken together, the results indicate that CK2a plays a very important role in human CRC cell proliferation and senescence CK2a knockdown or Zou et al Journal of Translational Medicine 2011, 9:97 http://www.translational-medicine.com/content/9/1/97 Page of 11 4.339, P < 0.05 for SW480 cells; Figure 5B) Accordingly, CK2a was positively correlated with CRC cell migration and invasion ability Table Correlation between the clinicopathological features and expression of the CK2a protein CK2a (%) Characteristics N Low expression P High expression Gender 0.646 Male 56 22 (39.3) 34 (60.7) Female Age 48 21 (43.8) 27 (56.2) ≥55 y 54 22 (40.7) 32 (59.3)